Tyrosine sulfation is an important post-translational modification that has been shown to significantly influence receptor/ligand interactions and modulate disease outcome in both plants and animals. Despite its importance, the precise role of tyrosine sulfation in protein function, host receptor activation and infection is not well understood. The long-term goal of this proposal is to elucidate the mechanisms with which tyrosine sulfation influences receptor-ligand interactions and resistance to infection. As a model for these studies, we will investigate the interaction of the rice XA21 immune receptor, a protein that is representative of a large class of plant and animal immune receptors, with the sulfated microbial protein, RaxX-sY, which is able to trigger a potent immune response in its host. We propose three complementary aims: Aim 1. Identify key amino acids that govern the interaction of sulfated RaxX with host receptors. Aim 2. Test if RaxB and CvaB are required for RaxX-sY processing and secretion. Aim 3. Determine the biological function of the Pald1 gene in the innate immune response. The proposed analyses will provide a framework for elucidating the critical role of sulfotyrosine in RaxX-sY/Xa21 interactions. To accomplish our goals, we will employ new experimental tools and approaches. These include using an expanded genetic code approach to produced full-length sulfated recombinant proteins in E. coli, new assays to assess receptor activation and ligand binding, and a liquid chromatography tandem mass spectrometry coupled with ultraviolet photodissociation to facilitate the characterization of sulfated tyrosine residues within peptides. The proposed studies are expected to lead to new insights regarding sulfated molecules and their interacting partners. The information gained from this research can be used to develop peptide-based reagents capable of inhibiting or activating receptor- ligand interactions with a high degree of affinity and specificity with potential applications in research, agriculture and medicine. Because sulfation of receptors or ligands is important in controlling the outcome of serious diseases and because innate immunity is critical for plant and animal defense against pathogens, the expected results will be broadly relevant to human health.